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imported>Xenbase gene generator |
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| '''Adapted By:''' P.Vize
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| '''The Plan:''' To link the Cy3 fluor onto tyramide. The reactive esters are effective couplers if they are in good condition. The reaction works best in an anhydrous environment- use fresh solvents.
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| '''The Materials:'''
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| Cy3 mono NHS ester Amersham PA13101 1 mg
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| tyramide Sigma T-2879 1 gm
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| dimethyl formamide Sigma T-8654
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| triethylamine Sigma T-0886
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| Ester stock Caution: NHS esters are very unstable. Once opened dissolve entire contents in DMF and use at once. If you cannot use all of the solution keep tightly sealed and store frozen in the dark- protect from absorbing water.
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| '''The solutions:'''
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| # Make a 10 mg/ml stock of your NHS ester in DMF (1 mg Cy3 NHS ester (MW = 766), 100 ul DMF) | |
| # Make DMF-TEA solution (1 ml DMF (dimethylformamide), 10 ul TEA (triethylamine))
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| # Make Tyramide solution (10 mg tyramide, 1 ml DMF-TEA)
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| '''The Reaction:'''
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| 100 ul Cy3 NHS in DMF
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| 33 ul tyramide solution
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| mix and incubate in dark at room temp for 2 hours.
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| add 1.2 ml ethanol , store in dark at 4 or -20
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| we have tried other ratios. although not the theoretical ratio this works very well to make good tyramide, we have repeated it from scratch twice and it is by far the best.
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| NHS-esters that didnt work...
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| Molecular Probes Alexa 633 failed, their TxRed failed too... They replaced both for free, but they failed again. We have tried multiple NHS : tyramide ratios (1.1:1, 1.5:1, 2:1) and still failed. We like the Piece and Amersham products. No company provided tyramides have worked either- including Oregon green, 633 and 646 from Molecular probes. We wasted over $1,000 on these. The Perkin-Elmer Cy5 does work but is really really weak and only works with strong antibodies, not for in situs.
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| '''Papers and other sites:'''
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| Davidson, L. A., and Keller, R. E. (1999). Neural tube closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation, and convergent extension. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10498689| Development, 126:4547-4556]
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| Zhou, X. and Vize, P.D. (2004). Proximo-distal specialization of epithelial transport processes within the Xenopus pronephric tubules. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15223337|Developmental Biology 271: 322-338.]
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| Davidson, Keller, and DeSimone (2004) Patterning and tissue movements in a novel explant preparation of the marginal zone of Xenopus laevis. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15183313| Gene Expression Patterns Jul;4(4):457-66.]
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| [http://www.faculty.virginia.edu/davidson/fluor_insitu/fluorescent_in_situ.html| Lance Davidson's flourescent in situ methods page]
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| Vize, P.D., McCoy, K.E., and Zhou, X. (2009). Multichannel wholemount fluorescent and fluorescent/chromogenic in situ hybridization in Xenopus embryos. [http://www.ncbi.nlm.nih.gov/pubmed/19498377| Nat Protoc. 4(6): 975-983.]
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